Human glucagon-like peptide-1 (GLP-1) is a 37 amino acid residue gastrointestinal hormone involved in the regulation of blood glucose metabolism, gastrointestinal secretion and metabolism and food intake. GLP-1 is originating from preproglucagon which is synthesized inter alia in the L-cells in the distal ileum, in the pancreas and in the brain. GLP-1 stimulates insulin secretion in a glucose-dependant manner, stimulates insulin biosynthesis, promotes beta cell rescue, decreases glucagon secretion, gastric emptying and food intake. Human GLP-1 is hydrolysed to GLP-1 (7-37) and GLP-1 (7-36)-amide which are both insulinotropic agents. Pharmacological doses of GLP-1 administered to type 2 diabetic patients have been shown to significantly raise circulating insulin levels and to lower plasma glucagon levels. The actions of GLP-1 are mediated by GLP-1 receptors in the pancreas, heart, kidney, central nervous system and gastrointestinal tract. Hence, GLP-1 is expected to become very important in the treatment of diabetes.
However, native human GLP-1 is rapidly inactivated by degradation by the plasma enzyme dipeptidyl peptidase IV (DPP-IV) to a truncated GLP-1(9-36)amide metabolite, which serves as a GLP-1 receptor antagonist. This confers the peptide with a short circulating half-life.
This short circulating half-life is a problem for many diabetes patients particularly in the type 2 diabetes segment who are subject to so-called “needle-phobia”, i.e. a substantial fear of injecting themselves. In the type 2 diabetes segment most patients are treated with oral hypoglycaemic agents. Since GLP-1 compounds are expected to be an injectable pharmaceutical product, the fear of injections may become a serious obstacle for the widespread use of these clinically very promising compounds.
Hence, a range of different approaches and methods have been used for modifying the structure of GLP-1 compounds in order to provide a longer duration of action in vivo. Thus, a considerable effort is e.g. being made to develop analogues and derivatives of GLP-1 compounds less susceptible to DPP-IV mediated hydrolysis in order to reduce the rate of degradation by DPP-IV. WO 2006/097538, WO 2006/097536, WO 2006/037810, WO2006/005667, WO2005/058958, WO 2005/027978, WO 98/08871 and US 2001/0011071 describe various GLP-1 analogues and derivatives, including GLP analogues comprising non-proteogenic amino acids (i.e. non-natural amino acids) which may confer a certain protection against hydrolysis by DPP-IV.
Polypeptides containing only proteogenic amino acids (i.e. natural amino acids) such as native GLP-1, can be produced using recombinant techniques or via chemical synthesis. However, polypeptides also containing non-proteogenic amino acids such as N-terminally extended GLP-1 analogues, cannot currently be prepared via recombinant expression techniques in a practical way and are in general prepared via chemical synthesis. The most widely used method for peptide synthesis is solid phase peptide synthesis where the adequate protected amino acids are incorporated in a stepwise manner using a polymer as a solid support.
Solid phase peptide synthesis (SPPS) can be very efficient in the preparation of some peptides, but the use of protected amino acids in combination with the consistent use of excess of reactants makes this approach relatively expensive. In addition, each amino acid prolongation in a solid phase polypeptide synthesis requires a thorough washing procedure. Typically, incorporation of one amino acid involves up to 10 washings steps with solvents like NMP, DMF or DCM.
When polypeptides, such as insulinotropic agents, GLP-1 analogues, truncated analogues of GLP-1 and derivatives of GLP-1 are synthesized using SPPS, the formation of secondary structures during the synthesis often leads to lower efficiency of the individual synthetic steps. As a consequence, larger peptides or peptides containing certain amino acid sequences are often produced in low purity and yields. The impurities are often deletion peptides where one or more amino acids are missing in the final sequence. These impurities can be very difficult to separate from the desired peptide, and result in a product contaminated with deletion peptides.
It is the aim of the present invention to provide an efficient and economic method for the preparation of GLP-1 analogues and derivatives which are DPP-IV protected by having non-proteogenic amino acids in the N-terminal part. The method combines the advantage of cost-efficient production of truncated GLP-1 precursor molecules, using recombinant techniques together with chemical synthesis of N-terminal extensions comprising non-proteogenic amino acids. A significant reduction of the cost of producing GLP-1 analogues or derivatives is achieved. Less expensive GLP-1 analogues and derivatives are highly desirable for maximizing the number of patients for whom treatment is available, as well as for exploiting the advantages of alternative delivery routes which have lower bioavailability than subcutaneous injection, e.g. transdermal and pulmonal delivery.